Esters are important flavor and fragrance compounds that are present in many food and beverage products. Many of these esters are produced by yeasts and bacteria during fermentation. While ester production in yeasts through the alcohol acyl transferase reaction has been thoroughly investigated, ester production through alcoholysis has been completely neglected. Here, we further analyze the catalytic capacity of the yeast Eat1 enzyme and demonstrate that it also has alcoholysis and thiolysis activities. Eat1 can perform alcoholysis in an aqueous environment in vitro, accepting a wide range of alcohols (C2-C10) but only a small range of acyl donors (C2-C4). We show that alcoholysis occurs in vivo in several Crabtree negative yeast species but also in engineered Saccharomyces cerevisiae strains that overexpress Eat1 homologs. The alcoholysis activity of Eat1 was also used to upgrade ethyl esters to butyl esters in vivo by overexpressing Eat1 in Clostridium beijerinckii. Approximately 17 mM of butyl acetate and 0.3 mM of butyl butyrate could be produced following our approach. Remarkably, the in vitro alcoholysis activity is 445 times higher than the previously described alcohol acyl transferase activity. Thus, alcoholysis is likely to affect the ester generation, both quantitatively and qualitatively, in food and beverage production processes. Moreover, mastering the alcoholysis activity of Eat1 may give rise to the production of novel food and beverage products.
Ethyl acetate is an industrially relevant ester that is currently produced exclusively through unsustainable processes. Many yeasts are able to produce ethyl acetate, but the main responsible enzyme has remained elusive, hampering the engineering of novel production strains. Here we describe the discovery of a new enzyme (Eat1) from the yeast Wickerhamomyces anomalus that resulted in high ethyl acetate production when expressed in Saccharomyces cerevisiae and Escherichia coli. Purified Eat1 showed alcohol acetyltransferase activity with ethanol and acetyl-CoA. Homologs of eat1 are responsible for most ethyl acetate synthesis in known ethyl acetate-producing yeasts, including S. cerevisiae, and are only distantly related to known alcohol acetyltransferases. Eat1 is therefore proposed to compose a novel alcohol acetyltransferase family within the alpha/beta hydrolase superfamily. The discovery of this novel enzyme family is a crucial step towards the development of biobased ethyl acetate production and will also help in selecting improved S. cerevisiae brewing strains.
A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain p-nitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 degrees C. The 1.4 A resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related alpha/beta hydrolase fold enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions.
TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100degC and pH 7.5. The positional specificity of TM0077 was investigated using 4-nitrophenyl--D-xylopyranoside monoacetates as substrates in a -xylosidase-coupled assay. TM0077 hydrolyzes acetate at positions 2, 3, and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine-substituted and native structures of TM0077 were determined at 2.1 and 2.5 resolution, respectively, revealing a classicalpha/beta-hydrolase fold. TM0077 assembles into a doughnut-shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride and paraoxon were determined to 2.4 and 2.1 , respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction.
Comparative analysis of the genome of the hyperthermophilic bacterium Thermotoga maritima revealed a hypothetical protein (EstA) with typical esterase features. The EstA protein was functionally produced in Escherichia coli and purified to homogeneity. It indeed displayed esterase activity with optima at or above 95 degrees C and at pH 8.5, with a preference for esters with short acyl chains (C2-C10). Its 2.6-A-resolution crystal structure revealed a classical alpha/beta hydrolase domain with a catalytic triad consisting of a serine, an aspartate, and a histidine. EstA is irreversibly inhibited by the organophosphate paraoxon. A 3.0-A-resolution structure confirmed that this inhibitor binds covalently to the catalytic serine residue of EstA. Remarkably, the structure also revealed the presence of an N-terminal immunoglobulin (Ig)-like domain, which is unprecedented among esterases. EstA forms a hexamer both in the crystal and in solution. Electron microscopy showed that the hexamer in solution is identical with the hexamer in the crystal, which is formed by two trimers, with the N-terminal domains facing each other. Mutational studies confirmed that residues Phe89, Phe112, Phe116, Phe246, and Trp377 affect enzyme activity. A truncated mutant of EstA, in which the Ig-like domain was removed, showed only 5% of wild-type activity, had lower thermostability, and failed to form hexamers. These data suggest that the Ig-like domain plays an important role in the enzyme multimerization and activity of EstA.
Carboxylic ester hydrolyzing enzymes constitute a large group of enzymes that are able to catalyze the hydrolysis, synthesis or transesterification of an ester bond. They can be found in all three domains of life, including the group of hyperthermophilic bacteria and archaea. Esterases from the latter group often exhibit a high intrinsic stability, which makes them of interest them for various biotechnological applications. In this review, we aim to give an overview of all characterized carboxylic ester hydrolases from hyperthermophilic microorganisms and provide details on their substrate specificity, kinetics, optimal catalytic conditions, and stability. Approaches for the discovery of new carboxylic ester hydrolases are described. Special attention is given to the currently characterized hyperthermophilic enzymes with respect to their biochemical properties, 3D structure, and classification.
Title: Characterization and structural modeling of a new type of thermostable esterase from Thermotoga maritima Levisson M, Van der Oost J, Kengen SW Ref: Febs J, 274:2832, 2007 : PubMed
A bioinformatic screening of the genome of the hyperthermophilic bacterium Thermotoga maritima for ester-hydrolyzing enzymes revealed a protein with typical esterase motifs, though annotated as a hypothetical protein. To confirm its putative esterase function the gene (estD) was cloned, functionally expressed in Escherichia coli and purified to homogeneity. Recombinant EstD was found to exhibit significant esterase activity with a preference for short acyl chain esters (C4-C8). The monomeric enzyme has a molecular mass of 44.5 kDa and optimal activity around 95 degrees C and at pH 7. Its thermostability is relatively high with a half-life of 1 h at 100 degrees C, but less stable compared to some other hyperthermophilic esterases. A structural model was constructed with the carboxylesterase Est30 from Geobacillus stearothermophilus as a template. The model covered most of the C-terminal part of EstD. The structure showed an alpha/beta-hydrolase fold and indicated the presence of a typical catalytic triad consisting of a serine, aspartate and histidine, which was verified by site-directed mutagenesis and inhibition studies. Phylogenetic analysis showed that EstD is only distantly related to other esterases. A comparison of the active site pentapeptide motifs revealed that EstD should be grouped into a new family of esterases (Family 10). EstD is the first characterized member of this family.
Title: Crystallization and preliminary crystallographic analysis of an esterase with a novel domain from the hyperthermophile Thermotoga maritima Sun L, Levisson M, Hendriks S, Akveld T, Kengen SW, Dijkstra BW, Van der Oost J Ref: Acta Crystallographica Sect F Struct Biol Cryst Commun, 63:777, 2007 : PubMed
A predicted esterase (EstA) with an unusual new domain from the hyperthermophilic bacterium Thermotoga maritima has been cloned and overexpressed in Escherichia coli. The purified protein was crystallized by the hanging-drop vapour-diffusion technique in the presence of lithium sulfate and polyethylene glycol 8000. Selenomethionine-substituted EstA crystals were obtained under the same conditions and three different-wavelength data sets were collected to 2.6 A resolution. The crystal belongs to space group H32, with unit-cell parameters a = b = 130.2, c = 306.2 A. There are two molecules in the asymmetric unit, with a V(M) of 2.9 A3 Da(-1) and 58% solvent content.
The hyperthermophilic euryarchaeon Pyrococcus abyssi and the related species Pyrococcus furiosus and Pyrococcus horikoshii, whose genomes have been completely sequenced, are presently used as model organisms in different laboratories to study archaeal DNA replication and gene expression and to develop genetic tools for hyperthermophiles. We have performed an extensive re-annotation of the genome of P. abyssi to obtain an integrated view of its phylogeny, molecular biology and physiology. Many new functions are predicted for both informational and operational proteins. Moreover, several candidate genes have been identified that might encode missing links in key metabolic pathways, some of which have unique biochemical features. The great majority of Pyrococcus proteins are typical archaeal proteins and their phylogenetic pattern agrees with its position near the root of the archaeal tree. However, proteins probably from bacterial origin, including some from mesophilic bacteria, are also present in the P. abyssi genome.
Pentameric ligand gated ion-channels, or Cys-loop receptors, mediate rapid chemical transmission of signals. This superfamily of allosteric transmembrane proteins includes the nicotinic acetylcholine (nAChR), serotonin 5-HT3, gamma-aminobutyric-acid (GABAA and GABAC) and glycine receptors. Biochemical and electrophysiological information on the prototypic nAChRs is abundant but structural data at atomic resolution have been missing. Here we present the crystal structure of molluscan acetylcholine-binding protein (AChBP), a structural and functional homologue of the amino-terminal ligand-binding domain of an nAChR alpha-subunit. In the AChBP homopentamer, the protomers have an immunoglobulin-like topology. Ligand-binding sites are located at each of five subunit interfaces and contain residues contributed by biochemically determined 'loops' A to F. The subunit interfaces are highly variable within the ion-channel family, whereas the conserved residues stabilize the protomer fold. This AChBP structure is relevant for the development of drugs against, for example, Alzheimer's disease and nicotine addiction.
The genome of the crenarchaeon Sulfolobus solfataricus P2 contains 2,992,245 bp on a single chromosome and encodes 2,977 proteins and many RNAs. One-third of the encoded proteins have no detectable homologs in other sequenced genomes. Moreover, 40% appear to be archaeal-specific, and only 12% and 2.3% are shared exclusively with bacteria and eukarya, respectively. The genome shows a high level of plasticity with 200 diverse insertion sequence elements, many putative nonautonomous mobile elements, and evidence of integrase-mediated insertion events. There are also long clusters of regularly spaced tandem repeats. Different transfer systems are used for the uptake of inorganic and organic solutes, and a wealth of intracellular and extracellular proteases, sugar, and sulfur metabolizing enzymes are encoded, as well as enzymes of the central metabolic pathways and motility proteins. The major metabolic electron carrier is not NADH as in bacteria and eukarya but probably ferredoxin. The essential components required for DNA replication, DNA repair and recombination, the cell cycle, transcriptional initiation and translation, but not DNA folding, show a strong eukaryal character with many archaeal-specific features. The results illustrate major differences between crenarchaea and euryarchaea, especially for their DNA replication mechanism and cell cycle processes and their translational apparatus.
